Stroke is the third leading cause of death and a major cause of long term disability in the US. Inadequate success of current thrombolytic therapy demonstrates a critical need for new targets to improve quality of life post-stroke. Neurons adapt to decreased oxygen delivery subsequent to stroke by up-regulating genes controlled by the hypoxia inducible factor (HIF). This transcription factor is comprised of two subunits: HIF1 alpha and HIF1 beta. Recent studies show that increased HIF1 during ischemia results in upregulation of apoptotic genes. Our preliminary data indicates that Transglutaminase 2 (TG2) binds to HIF1 beta resulting in attenuation of HIF activation and decreased cell death during ischemia. Also, TG2 inhibits the upregulation of the HIF controlled proapoptotic gene Bnip3, but not VEGF. Our hypothesis is that TG2 will facilitate neuronal protection following oxygen and glucose deprivation (OGD) by attenuating apoptotic genes induced by HIF1. We propose to delineate the effects of TG2 on HIF signaling and ischemic cell death with 3 aims. 1) To test the hypothesis that TG2 is upregulated during OGD and decreases HIF-dependent pro-apoptotic signaling. We will use a neuroblastoma cell line (SH-SY5Y) that overexpresses wild type TG2 and two TG2 constructs with point mutations that differentially affect 2 unique functions (transamidating and GTPase) of the enzyme. Using a HIF signaling reporter assay and analysis of mRNA and protein expression patterns of HIF dependent genes, we will distinguish which properties of TG2 are important for regulating HIF signaling. 2) To test the hypothesis that TG2 protects neuronal cells from ischemic insult and which functions of TG2 are important. Using SH-SY5Y cells and primary cortical neurons we will measure OGD-induced cell death and upstream apoptotic events. 3) To test the hypothesis that TG2 leads to overall protection in a mouse model of stroke. We will use a permanent Middle Cerebral Artery ligation model in C57BL/6 mice that express endogenous TG2, overexpress TG2, or lack TG2. We will measure infarct volumes using T2 weighted MRI as well as identifying genes regulated by TG2 with appropriate techniques. Our preliminary data show that overexpression of TG2 decreases stroke infarct volumes by 33%. We hypothesize that TG2 will lead to overall protection against stroke. [unreadable] [unreadable]